Identification of transcription factors involved in rice secondary cell wall formation

Using co-expression network analysis, we identified 123 transcription factors (TFs) as candidate secondary cell wall regulators in rice. To validate whether these TFs are associated with secondary cell wall formation, six TF genes belonging to the MYB, NAC or homeodomain-containing TF families were overexpressed or downregulated in rice. With the exception of OsMYB58/63-RNAi plants, all transgenic plants showed phenotypes possibly related to secondary cell wall alteration, such as dwarfism, narrow and dark green leaves, and also altered rice cinnamyl alcohol dehydrogenase 2 (OsCAD2) gene expression and lignin content. These results suggest that many of the 123 candidate secondary cell wall-regulating TFs are likely to function in secondary cell wall formation in rice. Further analyses were performed for the OsMYB55/61 and OsBLH6 TFs, the former being a TF in which the Arabidopsis ortholog is known to participate in lignin biosynthesis (AtMYB61) and the latter being one for which no previous involvement in cell wall formation has been reported even in Arabidopsis (BLH6). OsMYB55/61 and OsBLH6-GFP fusion proteins localized to the nucleus of onion epidermal cells. Moreover, expression of a reporter gene driven by the OsCAD2 promoter was enhanced in rice calli when OsMYB55/61 or OsBLH6 was transiently expressed, demonstrating that they function in secondary cell wall formation. These results show the validity of identifying potential secondary cell wall TFs in rice by the use of rice co-expression network analysis.